Heterocyclic compounds such as pyridine currently are recovered as constituents of coal tar, or are synthesized for example by the reaction of acetaldehyde with ammonia and formaldehyde to provide a pyridine, alpha-picoline and beta-picoline product mixture. Specialty heterocyclic aromatic chemicals are utilized in the production of adhesives, pesticides, vitamins, and the like.
Another prospective route to heterocyclic aromatic compounds is by the reaction of ammonia or a primary amine with a 2-hydroxymuconic semialdehyde to form a picolinic acid: ##STR1## Subsequent decarboxylation of the picolinic acid could provide the corresponding pyridines and substituted pyridines.
A potentially convenient source of 2-hydroxymuconic semialdehyde is by the microbiological oxidation of various hydrocarbon substrates. Microbiological oxidation of aromatic substrates is reviewed in Advances in Microbial Physiology, 6, 1-47 (1971); "Degradation Of Synthetic Organic Molecules In The Biosphere", pages 17-55, National Academy Of Sciences, 1972; and "Microbial Degradation Of Xenobiotics And Recalcitrant Compounds", pages 97-107, Academic Press, 1981. Strains of microorganisms are known which metabolize aromatic hydrocarbon substrates by the meta pathway via catechol and 2-hydroxymuconic semialdehyde to biomass and carbon dioxide.
Nature, 188, 560(1960) describes the cleavage of catechol with a new enzyme, catechol 2,3-oxygenase, to produce a product with a yellow color in the bioconversion medium. Ultraviolet absorption seems to indicate a 2-hydroxymuconic semialdehyde type product, which on standing with ammonium hydroxide appears to form alpha-picolinic acid.
Canadian Journal of Microbiology, 14, 1005(1968) describes the metabolism of p-xylene and m-xylene by species of Pseudomonas. A metabolite is produced which has an ultraviolet spectrum consistent with a 2-hydroxymuconic semialdehyde structure. Contact of the metabolite-containing solution with ammonium hydroxide yields a picolinic acid type product.
Biochemical Journal 106, 859(1968) also describes the formation of 5-methylpicolinic acid from 4-methylcatechol via 2-hydroxy-5-methylmuconic semialdehyde, utilizing a cell extract prepared from a microorganism grown on toluene sulfonate.
Journal of Bacteriology, 120(1), 31(1974) describes Pseudomonas putida mutants which metabolize phenol and cresols by the meta pathway via catechol and 2-hydroxymuconic semialdehyde intermediates. One mutant strain is described as being defective in both 2-hydroxymuconic semialdehyde hydrolase and dehydrogenase.
The potential of microbiological oxidation of an aromatic substrate such as toluene as a convenient source of 2-hydroxymuconic semialdehyde requires the construction of mutant strains of microorganisms which (1) metabolize an aromatic substrate via catechol or substituted catechol by means of the meta (catechol 2,3-oxygenase) pathway, and (2) allow the accumulation of 2-hydroxymuconic semialdehyde without its further assimilation to other metabolites.
Accordingly, it is an object of this invention to provide a process for construction of novel strains of microorganisms which metabolize catechol or a catechol-precursor by the meta pathway to accumulated 2-hydroxymuconic semialdehyde.
It is another object of this invention to provide a microbial culture which is capable of metabolizing toluene or substituted toluene to 2-hydroxymuconic semialdehyde or substituted 2-hydroxymuconic semialdehyde metabolite quantitatively, with an accumulation greater than about 0.1 gram of metabolite per liter of bioconversion medium.
Other objects and advantages of the present invention shall become apparent from the accompanying description and examples.